Zr-89-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs

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Zr-89-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid

organs

van der Veen, Elly L.; Giesen, Danique; Pot-de Jong, Linda; Jorritsma-Smit, Annelies; De

Vries, Elisabeth G. E.; Lub-de Hooge, Marjolijn N.

Published in:

Journal for immunotherapy of cancer DOI:

10.1136/jitc-2020-000938

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Publication date: 2020

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Citation for published version (APA):

van der Veen, E. L., Giesen, D., Pot-de Jong, L., Jorritsma-Smit, A., De Vries, E. G. E., & Lub-de Hooge, M. N. (2020). Zr-89-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs. Journal for immunotherapy of cancer, 8(2), [000938]. https://doi.org/10.1136/jitc-2020-000938

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89

_{Zr- pembrolizumab biodistribution is}

influenced by PD-1- mediated uptake in

lymphoid organs

Elly L van der Veen ,1 Danique Giesen,1 Linda Pot- de Jong,1

Annelies Jorritsma- Smit,2_{Elisabeth G E De Vries,}1_{Marjolijn N Lub- de Hooge} 2,3

To cite: van der Veen EL,

Giesen D, Pot- de Jong L,

et al. 89_{Zr- pembrolizumab}

biodistribution is influenced by PD-1- mediated uptake in lymphoid organs. Journal for ImmunoTherapy of Cancer

2020;8:e000938. doi:10.1136/

jitc-2020-000938

►Additional material is

published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ jitc- 2020- 000938).

Accepted 11 August 2020

1_{Department of Medical}

Oncology, UMCG, Groningen, Groningen, Netherlands

2_{Department of Clinical}

Pharmacy and Pharmacology, UMCG, Groningen, Groningen, Netherlands

3_{Department of Nuclear}

Medicine and Molecular Imaging, UMCG, Groningen, Groningen, Netherlands

Correspondence to

Dr Marjolijn N Lub- de Hooge; m. n. de. hooge@ umcg. nl © Author(s) (or their employer(s)) 2020. Re- use permitted under CC BY- NC. No commercial re- use. See rights and permissions. Published by BMJ.

ABSTRACT

Background To better predict response to immune

checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole- body distribution of zirconium-89 (89_{Zr) labeled programmed}

cell death protein-1 (PD-1) targeting pembrolizumab with positron- emission tomography (PET) in humanized mice.

Methods Humanized (huNOG) and non- humanized NOG

mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post 89_Zr-

pembrolizumab (10 µg, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a coinjection of excess (90 µg) unlabeled pembrolizumab or 89_{Zr- IgG}

4 control

(10 µg, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1.

Results PET imaging and biodistribution studies showed

high 89_{Zr- pembrolizumab uptake in tissues containing}

human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of 89_{Zr- pembrolizumab was}

lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative.

Conclusion 89_{Zr- pembrolizumab whole- body}

biodistribution showed high PD-1- mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of 89_{Zr- pembrolizumab whole- body distribution}

in patients.

BACKGROUND

Immune checkpoint inhibitors targeting the programmed cell death protein-1 (PD-1/ programmed death ligand-1 (PD- L1) pathway are showing impressive antitumor effects. However, not all patients respond and serious immune- related toxicity has been reported.1_{This has raised interest in}

better understanding the behavior of these drugs in the human body. PD- L1 and PD-1 are expressed by a broad range of immune cells, including T- cells, B- cells, natural killer (NK) cells, monocytes and dendritic cells. PD- L1 can be highly expressed by tumor cells, whereas PD-1 expression is most prominent in T- cells and lower in other immune cells.2

Biodistribution of PD-1 and PD- L1 targeting drugs will likely be influenced by the dynamic expression patterns of these targets.

Molecular imaging has proven to be an useful tool for studying drug biodistribu-tion.3 4_In_{table 1}_{, we summarized preclinical}

imaging studies that investigated biodistribu-tion of radiolabeled molecules targeting PD-1 and PD- L1.5–28_{Most studies that we reviewed}

focused on tracer distribution in the tumor and its microenvironment, without consid-ering PD-1 and PD- L1 expression in healthy immune tissues. Studies that do report on tracer uptake in lymphoid tissues are scarce and results are often limited to the spleen. Furthermore, most tracers targeting human PD-1/PD- L1 are not cross- reactive with murine proteins and relevant mouse models reconsti-tuted with (parts of) a human immune system are rarely used. A limited number of studies used NOD scid gamma (NSG) mice engrafted with human peripheral blood mononuclear cells (hNSG model).23–25 27_{The hNSG model}

has a high level of functional T- cells, however, it is also characterized by aberrant distribution of immune cells to murine immune tissues and other cell lineages remain underdevel-oped.29 Humanized mice that are engrafted with human CD34 + hematopoietic stem cells (HSCs) establish an immune- competent model with a broader set of developed human immune cells present and might therefore be a better surrogate for the human immune environment.

To gain more insight in the in vivo behavior of a human PD-1 targeting monoclonal

copyright.

on November 18, 2020 at University of Groningen. Protected by

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Table 1

Pr

eclinical imaging studies tar

geting

L1 and PD-1, using radiolabeled monoclonalantibody or small pr

oteins

Type of Imaging

Tracer

Origin and reactivity

Cr oss reactivity Animal model Tumor model Tracer dose

Imaging / biodistribution time point

Tumor uptake

Uptake lymphoid tissue

Ref L1 - antibodies SPECT/CT 111 L1.3.1 antibody

Murine anti- human

No

Balb/c nude mice 6 to 8 weeks old Immune deficient

Human br

east

cancer cell lines

1.5

µg (15.5 MBq) and

1.0

µg (10.0 MBq)

Imaging and ex vivo biodistribution at 24, 72 and 168

hours pi

32.8 (±6.8) %ID/g and 6.2 (±1.0) %ID/g at 168

hours pi for MB-231 and MCF-7 tumors r espectively L1 detection at dif fer ent expr ession levels in Br -3, SUM149 and BT474 tumors No ( 5 ) SPECT 111 DTP L1 antibody

Hamster anti- mouse

No

neu

- N transgenic mice

8 to 12 weeks old Immune competent NT2.5 (mouse mammary tumor) 7.4 MBq for imaging and 8.4

µg (0.93 MBq) for

biodistribution

Imaging on 1, 24, and 72 days pi and ex vivo biodistribution at 1, 24, 72, and 144

hours pi

Tumor uptake of 21.1 (±11.2) %ID/g at 144

hours

pi

Yes, spleen (63.5%±25.4 %ID/g) and thymus (16.8%±16.2 %ID/g) at 144

hours pi

Spleen uptake was blocked by coinjection of unlabeled antibody

( 6 ) SPECT 111 L1 antibody

Humanized anti- human

Cr

reactive

with mouse

NSG mice 6 to 8 weeks old Immune deficient

Human cell lines

100

µg (14.8 MBq) for

imaging and 8.5

µg (1.48

MBq) for biodistribution

Imaging and ex vivo biodistribution at 24, 48, 72, 96 and 120

hours pi

8.9 (±0.26) %ID/g at 72 hours pi for MDA- MB-231 tumors and 7.46 (±0.12) at 144

hours pi for H2444 tumors Detection of L1 at dif fer ent expr ession levels Yes, spleen (23.5±8.2) at 48 hours pi

Spleen uptake was blocked by

injection of unlabeled antibody ( 7 ) PET 64 L1 antibody

Cr

reactive

with mouse

Human cell lines

16.7 MBq (40

µg) for

imaging and 1.48 MBq (10

µg) for biodistribution

Imaging on 2, 24 and 48 hours pi and ex vivo biodistribution at 24 and 48 hours

pi

40.6 (±6.9) %ID/g, 17.2 (±2.1) %ID/g and 9.4 (±2.3) %ID/g at 48

hours pi for L1 positive CHO, MB-231

and SUM149 tumors respectively

High spleen uptake (~45 %ID/g) at 24

hours

pi after blocking with unlabeled antibody

(

8

)

Balb/c mice 4 to 6 weeks old Immune competent 4T1 (mouse mammary car

cinoma)

17.0 (±4.3) %ID/g at 48 hours pi for 4T1 tumors No high uptake observed in spleen (±12 %ID/g) and BA

T SPECT 111 DTP L1 antibody Rat mouse No

C57BL/6 mice 6 to 8 weeks old Immune competent B16F10 (murine melanoma) 15–16 MBq (60 µg) for imaging and 0.37 MBq (0.13 mg/kg) Imaging on 1, 24 and 72 hours pi and biodistribution at 1, 24, 72 and 96 hours pi

6.6 (±3.1) %ID/g at 24 hours pi for B16F10 tumors

Yes, spleen (47%±9.5 %ID/g) at 24

hours pi

and BA

T

Spleen uptake was blocked by coinjection of unlabeled antibody

( 9 ) PET 89Zr - L1 antibody Rat mouse No

C57BL/6 mice 6 to 8 weeks old Immune competent MEER (murine tonsil epithelium) or B16F10 (murine melanoma)

3.7 MBq (50

µg)

Imaging and ex vivo biodistribution at 48 and 96 hours

pi

Higher uptake in irradiated (20.1%±2.6 %ID/g) vs

irradiated

(11.1%±1.9

%ID/g) MEER tumors Higher uptake in irradiated (28.0%±4.9 %ID/g) vs

irradiated

(14.4%±1.4

%ID/g) B16F10 tumors

Yes, spleen (60% to 120%ID/g) and thymus (25% to 35%ID/g) Spleen uptake was blocked by pr

injection of unlabeled antibody ( 10 ) PET 89Zr - C4

(recombinant IgG1 antibody)

Engineer ed anti-human Cr reactive mouse

Nu/nu mice 3 to 5 weeks old Immune deficient H1975 and A549 (human NSCLC), PC3 (human prostatic small cell car

cinoma)

11.1 MBq for imaging and 1.85 MBq for ex vivo biodistribution Imaging and ex vivo biodistribution at 8, 24, 48, 72, 120

hours pi

~9 %ID/g~5 %ID/g and ~7 %ID/g at 48

hours pi

for H1975, A459 and PC3 tumors r

espectively

~13%

ID/g at 48

hours pi

for B16F16 tumors ~5 %ID/g tumor uptake at 48 hours pi in PDX model Detection of

induced

changes in

L1

expr

ession

Yes, spleen uptake of ~7 %ID/g and ~6 %ID/g at 48

hours pi in nu/nu and C57BL/6

mice r

espectively

Incr

eased uptake in the spleens of nu/nu

mice tr

eated with paclitaxel and spleens of

C57BL/6 mice tr

eated with doxorubicin

(

11

)

C57BL/6 mice 3 to 5 weeks old Immune deficient B16F10 (mouse melanoma)

Not r

eported

PDX model of EGFR mutant (L858R) NSCLC

Continued

copyright.

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Tracer

Tumor uptake

Ref SPECT/CT 111 L1 Rat murine No

Balb/c and C57BL/6 6 to 8 weeks old Immune competent

Murine cell lines

19.7 (±1.2) MBq (30

µg)

Imaging and ex vivo biodistribution at 72

hours

pi

~14.53 (±5.49) %ID/g,~16.29 (±5.57) %ID/g,~11.06 (±6.54) %ID/g,~14.94 (±4.01) %ID/g,~6.16 (±2.94) %ID/g, for Renca, 4T1, CT26, B16F1 and LLC1 respectively

Yes, spleen varying fr

om 13.09 %ID/g to

40.30 %ID/g. Thymus varying fr

om 6.09 %ID/g to 10.26 %ID/g ( 12 ) 111 L1

Murine anti- human

No humanized and humanized NSG mice MB-231 (human br east car cinoma) 11.9±1.6 MBq (1 µg) 111 L1 Or 11.5±0.4 MBq (2.8 µg) 111 contr ol mIgG1 LPS tr eatment 1 day befor e tracer injection

hours

pi

~40 %ID/g for non- humanized mice and ~60 %ID/g for humanized mice at 72

hours pi

~35 %ID/g for humanized mice after LPS tr

eatment at 72 hours pi ~8 %ID/g for 111 contr ol

mIgG1 in both non- humanized and humanized mice at 72

hours pi

Yes, spleen uptake ~20%

ID/g for

non-humanize mice and ~25%

ID/g for

humanized mice ~80 %ID/g for humanized mice after LPS treatment at 72

hours pi ~10 %ID/g for 111 contr ol mIgG1 (both gr oups 111 L1 Rat murine No

Balb/c and C57BL/6 6 to 8 weeks old Immune competent

Murine cell lines

Irradiation followed on day one by injection of 23.8±1.7

MBq

(30

µg)

hours

pi

Higher uptake in irradiated (26.3%±2.0 %ID/g) vs

irradiated

(17.1%±3.1

%ID/g) CT26 tumors Higher uptake in irradiated (15.7%±1.8 %ID/g) vs

irradiated (12.3%±1.7% ID/g) LLC1 tumors No dif fer ence uptake in

irradiated (14.9%±6.8 %ID/g) vs

irradiated

(16.7%±3.5%

ID/g)

for

B16F1 tumors

Spleen uptake ~14% to 17%ID/g for all models Higher uptake in lymph nodes of irradiated tumor models vs

irradiated tumor models L1 - small molecules PET 64 WL12 L1 binding peptide) Engineer ed anti-human No

High L1-expr essing CHO cell line

5.6 MBq for imaging and 1.5 MBq for ex vivo biodistribution Imaging and ex vivo biodistribution at 10

min, 0.5, 1 and 2 hour pi 14.9 (±0.8) at 1 hour pi in expr essing CHO tumors No ( 13 ) PET 18 NOT A-Z L1_ (anti L1

small molecule, affibody)

Engineer

ed

anti-human af

fibody

No

SCID beige mice 6 to 8 weeks old Immune deficient

IMVI

(human

melanoma) and SUDHL6 (human B- cell

lymphoma)

0.2 to 0.6 MBq Dynamic PET scan during 90 min 2.56 (±0.33) %ID/g at 90 min pi for LOX tumors

No ( 14 ) SPECT 99m L1 nanobodies Engineer ed mouse nanobodies Cr reactive human C57BL/6 mice (WT) vs CD8 depleted L1

KO mice 6 weeks old Immune competent TC-1 (mouse lung epithelial), WT TC-1

L1+ vs CRISPR/Cas9- modified TC-1 L1 KO 45 to 155 MBq (10 µg) nanobody Imaging 1 hour pi and

ex vivo biodistribution 80 min

pi

1.7 (±0.1) %ID/g for WT and 1.1 (±0.3) %ID/g for KO at 80

min pi

Yes, spleen 11.4 (±1.4) %ID/g for WT and 1.6±0.2%

ID/g for KO at 80

min pi

Lymph node uptake 3.5 (±0.8) %ID/g for WT and 0.4 (±0.1) %ID/g for KO at 80

min pi ( 15 ) PET 64 PD-1

ectodomain targeting

L1 Engineer ed anti-human Not specified

NSG mice Immune deficient CT26 (mouse colon cancer)

L1 (+) or L1(-) 8.5 MB (25 µg) Imaging at 1, 2, 4, and 24 hours. Ex vivo

biodistribution at 1 and 24 hours

~3 %ID/g for

L1 (+)

and ~1.8 %ID/g for

L1 (-) at

24

hours

pi

Yes, spleen ~5 %ID/g at 24

hours pi ( 16 ) Table 1 Continued Continued copyright.

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Tracer

Tumor uptake

Ref PET 18 BMS-986192 L1 small molecule) Engineer ed anti-human Af finity for

human & cynomolgus

L1,

no

binding to murine

L1)

Immune deficient mice

Human L2987 L1+) and HT -29 L1-) 5.6 MBq, block to 3 mg/kg Dynamic PET scan during 120

min

2.41 (±0.29) %ID/g for

L1 +and 0.82 (±0.11)

%ID/g for

L1-, 0.79

(±0.12) %ID/g after blocking in

L+

Yes, spleen uptake (no clear numbers)

( 17 ) Cynomolgus monkeys – 55.5 MBq

Dynamic PET scan during 150

min

–

Yes, spleen:muscle 12:1, after blocking spleen:muscle1.24:1

PET 64 PD-1 ectodomains (DOT A-/ NOT HAC, aglycosylated DOT A-/NOT A-HACA) Engineer ed anti-human Not specified

NSG mice 6 to 8 weeks old Immune deficient CT26 (mouse colon cancer)

L1(+)

or

L1(-)

0.7–3.7 MBq (10 to 15 µg) Imaging and ex vivo biodistribution at 1

hour pi

1.8 (±0.2) %ID/g for PD- L1(+) and 0.9 (±0.7) %ID/g

L1(-) for 64 NOT A- PD1 at 1 hour pi 4.2 (±0.8) %ID/g for L1(+) and 3.5 (±1.7) %ID/g for L1(-) for 64 NOT PD1 at 1 hour pi 2.7 (±1.1) %ID/g for L1(+) and 0.8 (±0.4) %ID/g for L1(-) for 64 NOT PD1 at 1 hour pi

Yes, spleen 4.0 (±3.1) %ID/g, 5.5 (±1.4) %ID/g and 1.4 (±0.4) %ID/g for 64 DOT A- PD1, 64 NOT PD1, and 64 Cu-NOT PD1 respectively ( 18 )

68Ga- PD-1 ectodomains (DOT

A-/ NOT HAC, aglycosylated DOT A-/NOT A-HACA) Engineer ed anti-human Not specified

NSG mice 6 to 8 weeks old Immune deficient CT26 (mouse colon cancer)

L1(+)

or

L1(-)

0.7 to 3.7 MBq (10 to 15 µg) Imaging and ex vivo biodistribution at 1

hour pi

3.8 (±1.6) %ID/g for PD- L1(+) and 1.7 (±1.3) %ID/g for

L1(-) for 68 NOT PD1 at 1 hour pi 2.8 (±1.5) %ID/g for L1(+) and 0.8 (±0.1) %ID/g for L1(-) for 68 DOT PD1 at 1 hour pi

Yes, spleen 3.5 (±0.6) %ID/g and 0.2 (±0.2) %ID/g for 68

NOT PD1 and 68 DOT A- PD1 respectively PET 64 FN3 L1

Small molecule

human No L1 3.7 (±0.4) MBq (8 to 10 µg) Imaging at 0.5, 1, 4, 18, and 24 hours pi followed by ex vivo biodistribution

5.6 (±0.9) %ID/g at 24 hours pi for CT26/

L1 tumors No ( 19 ) MB-231 (human br east cancer)

3.6 (±0.5) %ID/g at 24 hours pi for MDA- MB-231 tumors

PET 68 WL12 L1 binding peptide) Engineer ed anti-human No

Human cell lines

±7.4

MBq for imaging

and ±0.9

MBq for ex vivo

biodistribution

Imaging and ex vivo biodistribution at 15, 60, and 120

min pi

11.56 (±3.18) %ID/g, 4.97 (±0.8) %ID/g and 1.9 (±0.1) %ID/g for

L1,

MB-231

and SUM149 tumors respectively at 60

min pi No ( 20 ) PET 64 WL12 L1 binding peptide) Engineer ed anti-human No

NSG mice 5 to 6 weeks old Immune deficient Human cell lines: H226, HCC827,

L1+, hPDL1-, MDAMB231 ±7.4 MBq for imaging and ±0.74 MBq for ex vivo biodistribution

min

pi Treatment with atezolizumab

24

hours

prior to tracer injection (20

mg/kg)

~5.5 %ID/g,~8 %ID/g,~18 %ID/g,~5 %ID/g,~8 %ID/g for H226, HCC827, CHO- PDL1+,

PDL1- and MDAMB231 r espectively at 120 min pi Tr eatment r educed uptake

in all cell lines? T

umors

models?

Yes, spleen ~4 %ID/g, after tr

eatment ~3.5 %ID/g ( 21 ) PD1 - antibodies PET 64 PD-1 antibody

Hamster anti- mouse

No

Tr

eg+transgenic mice

(Foxp3+.LuciDTR) Immune competent B16F10 (mouse melanoma) 7.4 (±0.4) MBq (10– 12 µg) Blocking with fivefold molar excess Imaging and ex vivo biodistribution at 1

hour

,

24

hours, and 48

hours pi

7.4 (±0.71) %ID/g for

block vs 4.51 (±0.26)

%ID/g for blocking 48 hours

pi

Yes, spleen 23.04 (±4.97) %ID/g for non- block vs 14.39±0.53) %ID/g for blocking 48 hours

pi ( 22 ) Table 1 Continued Continued copyright.

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Tracer

Tumor uptake

Ref PET 89Zr - pembr olizumab

Not specified

NSG and humanized NSG mice (hNSG) A375 (human melanoma) 3.2 (±0.4) MBq (15 to 16 µg) Imaging at 1, 4, 18, 24, 48, 72, 96, 120 and 144

hours

pi, ex vivo biodistribution at 144

hours pi

1.8 (±0.4) %ID/g for NSG and 3.2 (±0.7) %ID/g for hNSG at 144

hours pi

Yes, spleen ~19 %ID/g for NSG and ~28 %ID/g for hNSG at 144

hours pi ( 23 ) 64Cu- pembr olizumab 7.4 (±0.4) MBq (20 to 25 µg) Imaging at 1, 4, 18, 24 and 48 hours pi, ex

vivo biodistribution at 48 hours

pi

5.7 (±0.6) %ID/g for NSG, 9.4 (±2.5) %ID/g for hNSG and 5.9 (±2.1) %ID/g for hNSG block at 48

hours pi

Yes, spleen ~6.5 %ID/g for NSG,~10.5 for hNSG, and ~7%

ID/g for hNSG block at 48 hours pi PET 89Zr - pembr olizumab

No

ICR (CD-1) mice and Hsd

Dawley

rats, 5 weeks old Immune competent

No tumor model Mice: 5 to 10 MBq (7 to 14 µg) Rats: 50 MBq (14 µg) Imaging at 3, 6, 12, 24, 48, 72, and 168 hours pi, ex vivo biodistribution at 168 hours pi No tumor model

Yes, spleen ~2.5 %ID/g for mice and ~1%

ID/g for rats 168

hours pi

(

24

)

NSG mice and humanized NSG mice engrafted with human PBMCs

SCID),

5–8 weeks old

No tumor model; PBMC engraftment

No tumor model

Yes, spleen ~8 %ID/g for NSG and ~4.5 %ID/g for

SCID) at 168 hours pi PET 89Zr - nivolumab

No

NSG mice and humanized NSG mice engrafted with human PBMCs

SCID

3–5 weeks old

A549 (human lung cancer)

5 to 10 MBq (7 to 14

µg)

Imaging at 3, 6, 12, 24, 48, 72, and 168

hours pi,

and ex vivo biodistribution at 168

hours pi.

3.88 (±0.38) %ID/g for NSG and 9.85 (±2.73) %ID/g for

SCID at

168

hours

pi

2.85 (±0.39) %ID/g for

PBL IgG contr

ol at

168

hours

pi

Yes, 7.48 (±0.47) %ID/g for NSG and 4.32 (±0.40) %ID/g for

SCID) at 168

hours

pi 3.05 (±0.79) %ID/g for

SCID IgG contr ol at 168 hours pi ( 25 ) PET 89Zr - nivolumab

Af finity for cynomolgus monkey Healthy human primates – 54.5 (±11.0) MBq (237 µg) Imaging at 24 hours, 96 hours, 144 hours and 192 hours – Yes, spleen at 192 hours SUV=17.63 Blocking 1 mg/kg at 192 hours SUV=2.5, 3 mg/kg SUV=2.62 ( 26 ) PET 64Cu- pembr olizumab

No

Humanized NSG mice

293T (human embryonic kidney cell line) expr

essing

L1

7.4 (±0.4) MBq (20 to 25 µg) Dynamic PET scans on 1, 2, and 4

hour pi during 3 min, at 18 and 24 hours pi during 5 min, at 24 hours pi during 10

min and at and

48

hours pi during 15

min

Ex vivo biodistribution at 1, 12, 24, and 48

hours pi

14.8 (±1.2) %ID/g for 293T tumors at 48

hours pi

0.44 (±0.01) %ID/g for A375 tumors at 48

hours pi

Yes, spleen (17.5%±1.6 %ID/g) at 48

hours

pi

(

27

)

A375 (human melanoma)

PDL1 + PD1 antibodies PET 64 PD-1 and 64 L1 antibody

Murine anti- mouse

No C57BL/6N mice deficient mice deficient mice Immune competent B16F10 (mouse melanoma) 1.13 (±0.31) MBq (1.5 µg) 64 PD-1 and 6.38 (±0.35) MBq (20 µg) 64 L1

Dynamic PET scan during 45–55 and 15–20

min at 24 hours pi for 64 Cu-PD-1 and 64 L1

respectively Ex vivo biodistribution at 48 hours

pi ±14 %IA/cm 3 in B16F10 tumor at 24 hours pi in vivo for 64 PD-1 and 64 L1 ±12 %IA/cm 3 in B16F10 tumor at 24 hours pi ex vivo for 64 L1

Yes, spleen (±20 %IA/cm

3) and lymph nodes

(20%–30%IA/cm

3) for 64

PD-1, spleen (15

%IA/cm

3), lymph nodes (7.5%–15%IA/cm 3) and BA T (±12 %IA/cm 3) for 64 L1

Detection of PD-1 +TILs after

py

L1 upr

egulation (mainly in lung) by

IFN-γ tr eatment visualized ( 28 ) WT ; type; AlF , aluminum fluoride; BA T, br

own adipose tissue; DOT

A,

tetraazacyclododecane- N,N',N

″,N'

″-tetraacetic acid ; DTP

A, diethylenetriaminepentaacetic acid; EGFR, epidermal gr

owth factor r

eceptor; %ID/g, per

centage of injected dose per gram;

IFN-γ, interfer

gamma; KO,

out; LPS,

lipopolysaccharide; NOT

A,

N,N',N''-triacetic acid; NSCLC,

small cell lung cancer; NSG, NOD SCID gamma; PBMC, peripheral blood mononuclear cell; PD-1, pr

ogrammed cell death pr

otein 1;

L1, pr

ogrammed

ligand 1; PDX,

derived xenograft; PET

, positr

on emission

tomography; pi,

injection; SPECT

, single photon emission CT

; TILs, tumor

- infiltrating lymphocytes.

Table 1

Continued

copyright.

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antibody (mAb), not cross- reactive with murine PD-1, we aimed to study the biodistribution of zirconium-89 (89_Zr)

radiolabeled pembrolizumab in melanoma- bearing humanized NOG mice (huNOG) engrafted with HSCs using positron- emission tomography (PET) imaging. To enable consecutive clinical translation of this approach, we developed and validated a good manufacturing prac-tices (GMP) compliant production process for 89_Zr-

pem-brolizumab. Finally, we put our data in perspective by summarizing results from current in vivo preclinical studies with PD-1 and PD- L1 targeting radiolabeled molecules.

METHODS Cell lines

The human melanoma cell line A375M was purchased from the American Type Culture Collection. Cell lines were confirmed to be negative for microbial contamina-tion and were authenticated on August 6, 2018, by Base-Clear using short tandem repeat profiling. A375M cells were routinely cultured in Roswell Park Memorial Insti-tute 1640 medium (Invitrogen) containing 10% fetal calf serum (Bodinco BV), under humidified conditions at 37°C with 5% CO2. Cells were passaged 1:10, twice a week. For in vivo experiments, cells in the exponential growth phase were used.

Development of 89_{Zr-pembrolizumab and}89_Zr-IgG

4

First, the buffer of pembrolizumab (25 mg/mL, Merck) was exchanged for NaCl 0.9% (Braun) using a Vivaspin-2 concentrator (30 kDa) with a polyethersulfon filter (Sartorius). Next, pembrolizumab was conjugated with the tetrafluorphenol- N- succinyldesferal- Fe(III) ester (TFP- N- sucDf; ABX) as described earlier, in a 1:2 TFP- N- sucDf:mAb ratio.30_{Conjugated product was purified from}

unbound chelator using Vivaspin-2 concentrators and stored at −80 °C. On the day of tracer injection, N- sucDf- pembrolizumab was radiolabeled with 89_{Zr, delivered}

as 89_{Zr- oxalate dissolved in oxalic acid (PerkinElmer),}

as described previously.30_{For in vivo studies,}

pembroli-zumab was radiolabeled at a specific activity of 250 MBq/ mg. IgG₄ control molecule (Sigma- Aldrich) was conju-gated with TFP- N- sucDf at a 1:3 molar ratio, followed by radiolabeling with 89_{Zr at similar specific activity of 250}

MBq/mg.

Quality control of 89_{Zr-pembrolizumab}

Size exclusion high- performance liquid chromatography (SE- HPLC) was used to determine the final number of TFP- N- sucDf ligands per antibody (chelation ratio). SE- HPLC analysis was also performed to assess potential aggrega-tion and fragmentaaggrega-tion for both N- sucDf- pembrolizumab and 89_Zr-_{pembrolizumab. An HPLC system (Waters)}

equipped with an isocratic pump (Waters), a dual wave-length absorbance detector (Waters), in- line radioactivity detector (Berthold) and a TSK- GEL G3000SWXL column (Tosoh Biosciences) was used with phosphate buffered

saline (PBS, sodium chloride 140.0 mmol/L, sodium hydrogen phosphate 0.9 mmol/L, sodium dihydrogen phosphate 1.3 mmol/L; pH 7.4) as mobile phase (flow 0.7 mL/min). Radiochemical purity of 89_Zr-

pembroli-zumab was measured by trichloroacetic acid precipita-tion assay.31_{Immunoreactivity of}89_{Zr- pembrolizumab was}

analyzed by a competition binding assay with unlabeled pembrolizumab. Nunc- immuno break apart 96- wells plates (Thermo Scientific) were coated overnight at 4°C with 100 µL of 1 µg/mL PD-1 extracellular domain (R&D Systems) in PBS, set to pH 9.6 with Na₂CO₃ 2M. Plates were washed with 0.1% Tween 80 in PBS and blocked for 1 hour at room temperature (RT) with 150 µL 1% human serum albumin (Albuman, Sanquin) in PBS. Multiple 1:1 mixtures of 89_{Zr- pembrolizumab with unlabeled}

pembroli-zumab were prepared, using a fixed concentration of

89_{Zr- pembrolizumab (7000 ng/mL) and varying}

concen-trations of unlabeled pembrolizumab (from 3.75 ng/mL to 12.5×106_{ng/mL). Of each mixture, 100 µL was added}

to the 96- wells plate and incubated for 2 hours at RT. After washing twice with washing buffer, radioactivity in each well was counted using a gamma counter (Wizard2_2480–

0019, SW 2.1, PerkinElmer). Counts were plotted against the concentration of competing unlabeled pembroli-zumab. The half maximal inhibitory concentration (IC₅₀) was calculated using GraphPad Prism 7 (GraphPad soft-ware). Immunoreactivity was expressed as the IC₅₀ value divided by the 89_Zr-_{pembrolizumab concentration to}

calculate the immune reactive fraction (IRF).

Animal studies

All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Groningen. Studies were performed in humanized NOG mice (NOD.Cg- Prkdcscid_Il2rgtm1Sug_{/JicTac, Taconic) and}

non- humanized NOG mice (Taconic) were used for control experiments. HuNOG mice are sublethally irra-diated 3 weeks after birth and subsequently reconstituted with human CD34+_{hematopoietic stem cells derived from}

fetal cord blood to express a functional human immune system including B- cells, T- cells, NK- cells, dendritic cells and monocytes. HuNOG and NOG mice were subcutane-ously xenografted with 5×106_{A375M human melanoma}

cells in 300 µL of a 1:1 mixture of PBS and Matrigel (BD Biosciences) on the right flank. Tumor growth was assessed by caliper measurements. When tumor volumes reached 100 to 200 mm3_{(after 2 weeks), 2.5 MBq}89_Zr-

pembroli-zumab (10 µg) was administered via retro- orbital injec-tion. Mice were anesthetized using isoflurane/medical air inhalation (5% induction, 2.5% maintenance).

The first group of huNOG mice received 10 µg

89_{Zr- pembrolizumab (n=5). In addition, a second group}

of huNOG mice xenografted with the same tumor model received a coinjection of 10 µg 89_{Zr- pembrolizumab and}

90 µg unlabeled pembrolizumab (n=4). To a third group of huNOG mice, 2.5 MBq 89_{Zr- IgG}

4 control (10 µg) was administered (n=4). Control NOG mice received 10 µg

89_{Zr- pembrolizumab (n=4).}

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PET imaging and ex vivo biodistribution

On day 7 post tracer injection (pi), PET scanning was performed. We selected this day based on optimal tumor- to- blood ratio and technical aspects, including feasible tracer specific activity and animal welfare. Mice were placed in a Focus 220 rodent scanner (CTI Siemens) on heating matrasses. Acquisition time was 60 min. A trans-mission scan of 515 s was performed using a 57_{Co point}

source to correct for tissue attenuation. After scanning, mice were sacrificed for ex vivo biodistribution. Bone marrow was collected from the femur bone by centrifugal- based separation. All other organs were dissected and counted in a gamma- counter (Wizard2_{2480–0019, SW 2.1,}

PerkinElmer). Tracer uptake in each organ was expressed as percentage of the injected dose per gram tissue weight, calculated by the following formula: %ID/g = (activity in tissue (MBq)/total injected activity (MBq))/tissue weight (g)×100. To compare ex vivo and in vivo uptake, ex vivo uptake was also calculated as mean radioactivity per gram tissue, adjusted for total body weight (SUV_mean_ex vivo), calculated with the following formula: SUVmeanexvivo = (activity in tissue (MBq)/total injected activity (MBq))×-mouse weight (g). Calculations are corrected for decay and background.

PET data was reconstructed and in vivo quantification was performed using PMOD software (V.4.0, PMOD tech-nologies LCC). Three- dimensional regions of interest were drawn around the tumor. For other organs and tissues, a size- fixed sphere was drawn in representative tissue parts. PET data was presented as mean standardized uptake value (SUV_mean _{in vivo}), calculated by the following formula: SUV_mean (g/mL) = (activity concentration (Bq/ mL)/applied dose (Bq))×weight (kg)×1000.

Autoradiography

Tumor and spleen from ex vivo biodistribution studies were formalin- fixed and paraffin embedded (FFPE). FFPE tissue blocks where cut into slices of 4 µM. These slices were exposed to a phosphor imaging screen (Perki-nElmer) for 72 hours and then scanned using a Cyclone phosphor imager (PerkinElmer).

Immunohistochemistry

Subsequent slices of the same tumor, spleen and mesen-teric lymph node tissue were stained for H&E, CD3, CD8 and PD-1. FFPE tumor, spleen and lymph node tissue were cut into 4 µm slices using a microtome (Microm Hm 355 s, Thermo Scientific) and mounted on glass slides. Tissue sections were deparaffinized and rehydrated using xylene and ethanol. Heat- induced antigen retrieval was performed in citrate buffer (pH=6) at 100°C for 15 min. Endogenous peroxidase was blocked by 30 min incuba-tion with 0.3% H₂O₂ in PBS. For CD3 staining, slides were incubated with rabbit anti- human CD3- antibody (Spring bioscience; clone SP162) in a 1:100 dilution in PBS/1% bovine serum albumin (BSA) at RT for 15 min. For CD8 staining, slides were incubated with rabbit anti- human CD8- antibody (Abcam; clone SP16) in a 1:50 dilution in

PBS/1% BSA at 4°C overnight. For PD-1 staining, slides were incubated with rabbit anti- human PD-1- antibody (Abcam, clone EPR4877(2)) in a 1:500 dilution in PBS/1% BSA at RT for 30 min. Human tonsil or lymph nodes tissues sections served ad positive control and were incubated with either CD3, CD8 or PD-1 antibody. As a negative control human tonsil or lymph nodes sections were incubated with rabbit IgG monoclonal antibody (Abcam, clone EPR25A) or PBS/1% BSA.

For CD3, CD8 and PD-1 staining, incubation with secondary antibody (anti- rabbit EnVision+_{, Dako) was}

performed for 30 min, followed by application of diam-inobenzidine chromogen for 10 min. Hematoxylin counterstaining was applied and tissue sections were dehydrated using ethanol and imbedded using mounting medium (Eukitt). H&E staining served to analyze tissue viability and morphology. Digital scans were acquired by a Nanozoomer 2.0- HT multi slide scanner (Hamamatsu).

89_{Zr-pembrolizumab manufacturing according to GMP}

To enable clinical application, GMP- compliant 89_Zr-

pem-brolizumab was developed. First, N- sucDf- pempem-brolizumab intermediate product was produced on a larger scale (60 mg batch, divided in 2.5 mg aliquots) and subse-quently radiolabeled with 89_{Zr, followed by purification,}

dilution and sterile filtration (online supplemental figure S1). Release specifications were defined, as shown in online supplemental table S1. All analytical methods for quality control (QC) were validated. According to protocol validation of both N- sucDf- pembrolizumab and

89_{Zr- pembrolizumab, manufacturing consisted of three}

independent validation runs, including complete release QC. Stability of N- sucDf- pembrolizumab stored at −80 °C was studied up to 6 months and stability of 89_Zr-

pembroli-zumab was determined up to 168 hours at 2°C to 8°C stored in a sterile, type 1 glass injection vial. In addition, in use stability was demonstrated at RT in a polypropylene syringe for up to 4 hours (online supplemental table S2).

Statistical analysis

Data are presented as median±IQR. A Mann- Whitney U test, followed by a Bonferroni correction was performed to compare groups (GraphPad, Prism 7). P values ≤0.05 were considered significant. If not indicated otherwise, results were not statistically significant.

RESULTS

89_{Zr-pembrolizumab development for in vivo studies}

We optimized the conjugation processes of pembroli-zumab with the TFP- N- sucDf chelator and its subsequent radiolabeling with 89_{Zr. For in vivo studies, N-}_sucDf-

pembrolizumab was produced with >60% yield and average 1.7 chelators per antibody (online supplemental figure S2, table S1). N- sucDf- pembrolizumab was subsequently radiolabeled with 89_{Zr at a specific activity of 250 MBq/}

mg, with radiochemical purity of >95% after purification. Both N- sucDf- pembrolizumab and 89_{Zr- pembrolizumab}

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were stable, as shown in online supplemental table S1, S2 and figure S2. Immunoreactivity was not impaired by conjugation or radiolabeling.

89_{Zr-pembrolizumab imaging and biodistribution in humanized}

mice

PET imaging revealed 89_Zr-_{pembrolizumab uptake in}

tumor, but also in healthy tissues, including liver, spleen and lymph nodes, of A375M tumor- bearing huNOG mice (figure 1A,B). Consistent with these results, ex vivo biodis-tribution at day 7 pi showed highest 89_{Zr- pembrolizumab}

uptake in spleen (SUV_mean 30.5, IQR 15.8 to 67.7), mesen-teric lymph nodes (SUV_mean 20.4, IQR 8.0 to 25.2), bone marrow (SUV_mean 14.5, IQR 6.1 to 32.8), thymus (SUV_mean 1.3, IQR 1.1 to 2.1), liver (SUV_mean, IQR 6.0, IQR 3.4 to 9.9) and tumor (SUV_mean 5.1, IQR 3.3 to 8.9) (figure 1C, online supplemental table S3).

Tumor uptake of 89_{Zr- pembrolizumab was variable and}

slightly higher than tumor uptake observed for 89_{Zr- IgG} 4 control, however not significant due to small groups of mice (SUV_mean 5.1, IQR 3.3 to 8.9 vs SUV_mean 3.5, IQR 2.7 to 4.4) (figure 1C). This may be explained by low PD-1 expression found in all tumors by immunohistochemical

(IHC) analysis (figure 2). 89_{Zr- pembrolizumab tumor-}

to- blood ratio also did not differ from 89_{Zr- IgG}

4 control (figure 1D).

89_{Zr- pembrolizumab in huNOG mice showed higher}

uptake in lymphoid tissues compared with 89_{Zr- IgG} 4 control: spleen (SUVmean 13.9, IQR 7.1 to 21.4, NS, p=0.254), mesenteric lymph nodes (SUV_mean 2.3, IQR 1.4 to 4.4, NS, p=0.114), salivary gland (SUVmean 2.1, IQR 1.2 to 2.9, NS, p=0.635), bone marrow (SUV_mean 8.8, IQR 7.6 to 10.0, NS, p=1.714) and thymus (SUV_mean 0.5, IQR 0.4 to 1.1, p=0.1714), indicating that 89_{Zr- pembrolizumab}

uptake in these tissues is, at least partly, PD-1- mediated.

89_{Zr- pembrolizumab tissue- to- blood (T:B) and tissue- to-}

muscle (T:M) ratios in lymphoid organs confirmed high uptake in these tissues (figure 1D,E). Additionally, rela-tively high 89_{Zr- IgG}

4 uptake was found in spleen, bone marrow and liver compared with other organs, suggesting

89_{Zr- pembrolizumab uptake in these tissues is also due}

to Fcγ receptor (FcγR)- binding of the antibody’s Fc- tail. High 89Zr- IgG4 uptake was less evident in lymph nodes and thymus.

Figure 1 In vivo PET imaging and ex vivo biodistribution of 89_{Zr- pembrolizumab in immunocompetent humanized NOG}

mice. Mice were xenografted with A375M tumor cells and received tracer injection at day 0. For blocking studies huNOG mice received a 10- fold excess of unlabeled pembrolizumab (huNOG excess). As a control for non- specific uptake huNOG mice were injected with 89_{Zr- IgG}

4. PET imaging performed on day 7 post injection (pi). (A) In vivo PET examples (maximum intensity

projections) at day 7 pi showing uptake in tumor (T), axillary lymph nodes (LN), liver (L) and spleen (S). (B) In vivo uptake of

89_{Zr- pembrolizumab in spleen, lymph nodes (axillary), liver and tumor, at day 7 pi. Uptake is expressed as SUV}

mean. (C) Ex vivo

biodistribution of 89_{Zr- pembrolizumab in humanized NOG mice. Uptake is expressed as mean radioactivity per gram tissue,}

adjusted for total body weight (SUV_mean_{ex vivo}). Data expressed as median±IQR *p≤0.05. BAT, brown adipose tissue; huNOG, humanized NOG mice; MLN, mesenteric lymph nodes; PET, positron emission tomography.

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89_{Zr- pembrolizumab spleen uptake in huNOG mice}

was blocked by the addition of a 10- fold excess unlabeled pembrolizumab (SUV_mean 30.5, IQR 15.8 to 67.7 versus SUV_mean 5.1, IQR 4.3 to 7.0, p=0.032) (figure 1B,C). Uptake in other lymphoid organs and liver was also reduced by addition of unlabeled mAb dose, whereas uptake in non- lymphoid tissues was unaffected (online supplemental table S3). Tracer activity in blood pool was increased by addition of unlabeled mAb (SUV_mean 0.1, IQR 0.0 to 1.8 to SUV_mean 2.2, IQR 1.4 to 7.4), but uptake in tumor did not change.

Autoradiography confirmed PET imaging results on a macroscopic level, showing high uptake in spleens of huNOG mice compared with spleens of mice that received an additional unlabeled pembrolizumab dose (figure 3). Furthermore, comparable tumor uptake was found for different dose groups. IHC analysis on spleen and lymph node tissue of huNOG mice revealed that PD-1, CD3 and CD8 positive cells were present. CD3 and CD8 cells were also present in tumor tissue of huNOG mice (figure 2), however, PD-1 staining of these tumors was negative.

89_{Zr- pembrolizumab biodistribution in NOG control}

mice clearly showed a different pattern than in huNOG mice, with high uptake in liver (SUV_mean 16.9, IQR 5.1 to 26.2) and spleen (SUV_mean 49.6, IQR 16.6 to 135.6),

whereas 89_{Zr- pembrolizumab tumor uptake in NOG mice}

was similar to huNOG mice (SUV_mean 9.3, IQR 4.5 to 15.7 vs SUV_mean 5.1, IQR 3.3 to 8.9) (online supplemental figure S3). High 89_Zr-_{pembrolizumab spleen uptake}

in this model may be unexpected, since limited T- cells are present in NOG mice (online supplemental figure

Figure 2 IHC analysis of spleen, mesenteric lymph node and tumor tissue humanized NOG mice. Formalin- fixed and paraffin

embedded tissue blocks where cut into slices of 4 µM and stained for PD-1, CD3 and CD8 (40x). H&E staining served to analyze tissue viability and morphology (40x). Scalebar: 50 µm. IHC, immunohistochemical; PD-1, programmed cell death protein-1.

Figure 3 Autoradiography of spleen and tumor tissue

humanized NOG mice (huNOG). Formalin- fixed and paraffin embedded tissue blocks where cut into slices of 4 µM. These slices were exposed to a phosphor imaging screen for 72 hours and were then scanned using a Cyclone phosphor imager.

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S3). However, high spleen uptake in severely immuno-compromised mice has been described previously and is potentially Fcγ receptor- mediated.23 24 32_Moreover,

spleen weights in NOG mice were lower than in huNOG mice (NOG: 0.017 g±0.015 g; huNOG: 0.037 g±0.016 g, p=0.036), which resulted in higher tracer uptake expressed as %ID per gram spleen tissue for NOG mice. A low spleen weight may result from high radiosensitivity of NOG splenocytes, which can lead to toxicity.33

Critical steps in 89_{Zr-pembrolizumab manufacturing}

The production processes for N- sucDf- pembrolizumab intermediate product and 89_{Zr- pembrolizumab for in vivo}

studies were modified to comply with GMP requirements. In the conjugation reaction, pH is increased from 4.5 to 8.5, performed in small titration steps, as described earlier by Verel et al.30_{During this pH transition, precipitation}

occurred at 6.5 to 7.0, which was re- dissolved at pH >7.5. No precipitation was observed when pH was changed abruptly, for example, by buffer exchange, to pH 8.5 during conjugation and to pH 4.5 for removal of Fe(III). This indicates potential instability of pembrolizumab at pH 6.5 to 7.0. Formation of aggregates may be explained by the fact that pembrolizumab is an IgG₄ type mAb, which forms non- classical disulfide bonds. In contrast, IgG₁ type antibodies can only form classical disulfide bonds. There are many other determinants of antibody stability besides disulfide bond formation, however, this phenomenon was not seen previously with the radiolabeling of IgG₁ type antibodies.31 33 34

Immunoreactivity was not affected when pembroli-zumab showed precipitation during pH transition, demonstrated by comparable IRF for precipitated N- sucDf- pembrolizumab and for non- precipitated N- sucDf- pembrolizumab (online supplemental figure S4). However, it is unknown whether the pembrolizumab struc-ture is modified by the formation of precipitates. There-fore, the method for pH transition by buffer exchange was incorporated in the conjugation protocol for pembroli-zumab. Production of clinical grade 89_{Zr- pembrolizumab}

was performed as previously described by Verel et al.30

89_{Zr-pembrolizumab GMP validation}

Three consecutive batches of conjugated and radiola-beled pembrolizumab were produced at clinical scale and complied with all release specifications (online supplemental tables S1 and S2), indicating that our process for manufacturing clinical grade 89_Zr-

pembroli-zumab is consistent and robust. 89_{Zr- pembrolizumab}

was obtained with a specific activity of 37 MBq/mg and mean IRF of 1.35±0.6 (n=3). Stability studies revealed that N- sucDf- pembrolizumab remained compliant to release specifications up to 6 months storage at −80°C, therefore N- sucDf- pembrolizumab shelf- life was set at 6 months. Stability studies are ongoing and shelf- life may be extended if future time points remain within spec-ifications. Data obtained during process development and validation were used to compile the investigational

medicinal product dossier (IMPD), which includes all information regarding quality control, production and validation of 89_{Zr- pembrolizumab. Based on this IMPD,} 89_{Zr- pembrolizumab has been approved by competent}

authorities for use in clinical studies.

DISCUSSION

This study reveals 89_{Zr- pembrolizumab whole- body}

distri-bution in tumor- bearing huNOG mice established with a broad set of developed immune cells. Tumor uptake of

89_{Zr- pembrolizumab was markedly lower than uptake in}

lymphoid tissues such as spleen, lymph nodes and bone marrow, but higher than uptake in other organs. Impor-tantly, high uptake in lymphoid tissues could be reduced with a 10- fold excess of unlabeled pembrolizumab. This contrasts with 89_{Zr- pembrolizumab tumor uptake,}

which was not reduced by the addition of unlabeled pembrolizumab.

Our study nicely shows the in vivo behavior of 89_Zr-

pem-brolizumab, which, apart from IgG pharmacokinetics determined by its molecular weight and Fc tail, is predom-inantly driven by its affinity for PD-1 (Kd:~30 pM). The PD-1 cell surface receptor is primarily expressed on acti-vated T- cells and pro B- lymphocytes, which are abun-dantly present in our huNOG mouse model. Lymphocytes are highly concentrated in organs that are key players of the immune system: lymph nodes, spleen, thymus, bone marrow as well as tonsils, adenoid and Peyer’s patches. From our PET imaging and ex vivo biodistribution data, we learned that 89_{Zr- pembrolizumab distributed mainly}

to lymphoid organs, where PD-1 expressing immune cells are present.

89_{Zr- pembrolizumab showed relatively low and variable}

tumor uptake, however, this uptake could be visualized with PET imaging 7 days pi and was higher than in non- lymphoid tissues. We hypothesized there may be PD-1- mediated 89Zr- pembrolizumab tumor uptake, but we also found tumor uptake for 89Zr- IgG4, suggesting part of the 89_{Zr- pembrolizumab tumor uptake is FcγR- mediated. In}

our mouse model, few PD-1 positive immune cells may have traveled to the tumor, thereby potentially limiting

89_Zr-_{pembrolizumab tumor uptake. Interestingly, the}

addition of unlabeled pembrolizumab did not influence tumor uptake. This is likely caused by substantial increase of 89_{Zr- pembrolizumab in blood pool as a direct}

conse-quence of adding excess unlabeled pembrolizumab, warranting a continuous pembrolizumab supply to the tumor.

Ex vivo immunohistochemical analysis revealed CD3 and CD8 positive lymphocytes were present in tumor, but limited PD-1- expression was found. Immune checkpoint protein expression status in tumor- infiltrating lympho-cytes is highly dynamic.35 36_{This so-}_{called ‘immune}

phenotype’ depends on several factors, including tumor type, location and mutational burden. Our results indi-cate that, whereas PD-1 expression may demonstrate large

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variation, 89_{Zr- pembrolizumab PET imaging is able to}

capture PD-1 dynamics in both tumor and healthy tissues. Compared with earlier preclinical studies with radio-labeled pembrolizumab in the hNSG model, we found higher 89_{Zr- pembrolizumab uptake in spleen and other}

lymphoid tissues.23 24_{This likely reflects the presence of}

multiple hematopoietic cell lineages, including B- cells, T- cells, NK- cells, dendritic cells and monocytes, and thus higher PD-1 expression, in our huNOG model compared with the hNSG model. Molecular imaging studies with radiolabeled antibodies generally show distribution to the spleen. It also known that Fc/FcγR- mediated immu-nobiology of the experimental mouse model plays a key role in the in vivo biodistribution and tumor targeting.33

In our mouse model, we also observed 89_{Zr- IgG}

4 uptake in lymphoid tissues, indicating 89_{Zr- pembrolizumab uptake}

in these organs may have an FcγR- mediated component. For most radiolabeled antibodies without an immune target, spleen uptake in patients is ~5 %ID/kg.37_This

supports the idea that, independent of their target, anti-bodies often show distribution to the spleen. However, spleen uptake may be higher if PD-1 or PD- L1 is present.

Pembrolizumab has an IgG₄κ backbone with a stabi-lizing SER228PRO sequence alteration in the Fc- region to prevent the formation of half molecules. The IgG₄ backbone of pembrolizumab may slightly differ from the IgG₄ control molecule that we used for our experiments, however, FcγR- binding affinity and kinetics of pembroli-zumab appears to be very similar to IgG₄.38_{We, therefore,}

consider the used IgG₄ control molecule to provide a useful indication of the extent of FcγR- mediated uptake. In this respect, FcγR- mediated uptake may be present in the spleen but potentially also in liver and tumor, since these tissues demonstrate relatively high uptake of

89_{Zr- IgG} 4.

PD-1 is predominantly expressed on activated T- cells while its ligand PD- L1 is expressed by a broader range of immune cells as well as tumor cells. It is therefore to be expected that biodistribution of antibody tracers targeting PD- L1 may deviate from the biodistribution results that we described here for 89_{Zr- pembrolizumab. In}_{table 1}_{, we}

presented an overview of preclinical imaging and biodis-tribution studies using anti- PD-1 and anti- PD- L1 tracers. Data turned out to be highly variable, mostly focused on tumor and not on the immune system, and therefore not just comparable. From our results, we increasingly realize that it is extremely important for interpretation of these type of data to know the characteristics of the anti-body (origin, cross- reactivity, Fc- backbone, target, target- affinity and dose), the animal model (mouse strain, age, immune status and tumor cell line) and time points, vari-ables we detailed in the table.

As for preclinical studies, data on the distribution of PD-1 and PD- L1 targeting antibodies to lymphoid organs in patients is still limited. A clinical imaging study in 13 patients demonstrated modest 89_{Zr- nivolumab spleen}

uptake of SUV_mean 5.8±0.7, whereas uptake of this radiola-beled antibody targeting PD-1 in other lymphoid tissues

was not addressed.39 89_{Zr- atezolizumab ( PD- L1}

anti-body) imaging in 22 patients revealed spleen uptake with an SUV_mean of 15. 89_{Zr- atezolizumab also distributed to}

other lymphoid tissues and sites of inflammation, whereas uptake in non- lymphoid organs was low. The high spleen uptake could at least partly be explained by presence of PD- L1 in endothelial littoral cells of the spleen.40 To perceive what can be expected for 89_{Zr- pembrolizumab}

PET imaging in patients, how results may be interpreted and potentially translated to predicting response, knowl-edge on which immune cells express PD-1 and where these cells are located in the human body is of utmost importance.

With our study, we validated the use of 89_Zr-

pembroli-zumab PET imaging to evaluate PD-1- mediated uptake in tumor and immune tissues in a setting that allowed for comparing tracer uptake and whole tumor tissue analysis. To enable evaluation of 89_{Zr- pembrolizumab}

biodistribu-tion in humans, we developed clinical grade 89_Zr-

pem-brolizumab. Clinical 89_{Zr- pembrolizumab PET imaging}

in patients with melanoma and NSCLC before treatment with pembrolizumab is currently performed at our center ( ClinicalTrials. gov Identifier NCT02760225), and may elucidate if tracer tumor uptake correlates to response and if uptake in healthy PD-1 expressing tissues correlates to toxicity.

CONCLUSION

We demonstrated the in vivo biodistribution of 89_Zr-

pem-brolizumab in humanized mice, and found uptake in tumor with the highest uptake in the lymphoid system, reflecting the presence of PD-1. Insight in the in vivo behavior and biodistribution of immune checkpoint targeting monoclonal antibodies might aid in better understanding immune checkpoint inhibition therapy and could potentially help explaining variation in response as well as potential toxicity due to uptake in healthy (immune) tissues.

Twitter Elisabeth G E De Vries @VriesElisabeth

Contributors ELvdV was involved in project design and conceptualization, was involved in tracer development and GMP validation, wrote the IMPD, performed animal studies, performed ex vivo analyses, data analysis and wrote the manuscript; DG was involved in study conceptualization, data analysis, performed ex vivo analyses and wrote the manuscript; LPdJ was involved in tracer development and GMP validation, performed animal studies, performed ex vivo analyses and edited the manuscript; AJS was involved in GMP validation, wrote the IMPD and edited the manuscript; EGEdV was involved in project design and conceptualization, supervised the study and edited the manuscript; MNLdH was involved in project design and conceptualization, supervised the study and edited the manuscript. All authors read and approved the final manuscript.

Funding The research leading to these results received funding from the Innovative Medicines Initiatives 2 Joint Undertaking under grant agreement No 116106 (TRISTAN). This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA.

Competing interests EGEdV reports grants from IMI TRISTAN (GA no.116106), during the conduct of the study; consulting and advisory role for NSABP, Daiichi Sankyo, Pfizer, Sanofi, Merck, Synthon Biopharmaceuticals; grants from Amgen, Genentech, Roche, Chugai Pharma, CytomX Therapeutics, Nordic Nanovector,

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G1 Therapeutics, AstraZeneca, Radius Health, Bayer, all made available to the institution, outside the submitted work.

Patient consent for publication Not required.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement Data are available upon reasonable request. The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. See http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.

ORCID iDs

Elly L van der Veen http:// orcid. org/ 0000- 0001- 6224- 8114

Marjolijn N Lub- de Hooge http:// orcid. org/ 0000- 0002- 5390- 2791

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